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The Journal of Immunology, 2002, 169: 1159-1167.
Copyright © 2002 by The American Association of Immunologists

Generation of CD4+CD45RA+ Effector T Cells by Stimulation in the Presence of Cyclic Adenosine 5'-Monophosphate- Elevating Agents1

Ana Suárez*, Lourdes Mozo{dagger} and Carmen Gutiérrez2,*,{dagger}

* Department of Functional Biology, Area of Immunology, Universidad de Oviedo, and {dagger} Department of Immunology, Hospital Central de Asturias, Oviedo, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
After TCR cross-linking, naive CD4+CD45RA+ T cells switch to the expression of the CD45RO isoform and acquire effector functions. In this study we have shown that cAMP-elevating agents added to anti-CD3- and anti-CD28-stimulated cultures of T lymphocytes prevent acquisition of the CD45RO+ phenotype and lead to the generation of a new subpopulation of primed CD4+CD45RA+ effector cells (cAMP-primed CD45RA). These cells displayed a low apoptotic index, as the presence of dibutyryl cAMP (dbcAMP)-rescued cells from CD3/CD28 induced apoptosis. Inhibition of CD45 splicing by dbcAMP was not reverted by addition of exogenous IL-2. cAMP-primed CD45RA cells had a phenotype characteristic of memory/effector T lymphocytes, as they showed an up-regulated expression of CD2, CD44, and CD11a molecules, while the levels of CD62L Ag were down-regulated. These cells also expressed the activation markers CD30, CD71, and HLA class II Ags at an even higher level than CD3/CD28-stimulated cells in the absence of dbcAMP. In agreement with this finding, cAMP-primed CD45RA cells were very efficient in triggering allogenic responses in a MLR. In addition, cAMP-primed CD45RA cells produce considerable amounts of the Th2 cytokines, IL-4, IL-10, and IL-13, whereas the production of IFN-{gamma} and TNF-{alpha} was nearly undetectable. The elevated production of IL-13 by neonatal and adult cAMP-primed CD45RA cells was specially noticeable. The cAMP-dependent inhibition of CD45 splicing was not caused by the production of immunosuppressor cytokines. These results suggest that within the pool of CD4+CD45RA+ cells there is a subpopulation of effector lymphocytes generated by activation in the presence of cAMP-elevating agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human mature CD4+ T cells are functionally heterogeneous and can be divided into naive and memory/effector populations based on the expression of cell surface markers. Although the most significant differences between these two populations are functional, the leukocyte common Ag (CD45) was promoted as a potential marker of memory T cells (1, 2). CD45 Ag may be found in various isoforms depending on alternative splicing of three extracellularly expressed exons. The expression of the highest molecular weight isoform, referred to as CD45RA, defines the population of CD4+ unprimed lymphocytes. Activation of naive lymphocytes through TCR engagement is followed by loss of CD45RA Ag and transition to the expression of the smallest molecular weight isoform CD45RO (3, 4). These primed cells express high levels of activation Ags, acquire effector functions, and need less costimuli requirements for further activation. In addition, naive and memory/effector T cells display different patterns of adhesion molecules, reflecting different migratory pathways (5, 6). One of the most striking functional differences between naive and memory/effector CD4+ T cells is the ability of the latter to secrete the full range of T cell cytokines and differentiate to Th1 and Th2 subsets (7, 8).

More recently, the use of the CD45 isoform expression as the unique marker of memory has been questioned by various studies which suggest that the CD45RA Ag may be re-expressed by previously activated CD4+ and CD8+ T cells (revertant CD45RO) (9, 10). On the other hand, naive T cells may be activated by cytokines leading to the expression of surface molecules characteristic of memory cells while remaining as CD45RA+ (11, 12, 13). It has been suggested that the proportion of CD45RA+ T cells in adult blood expressing increased levels of adhesion molecules may be revertant CD45RO or cytokine-expanded cells (14, 15).

Intracellular cAMP is an important physiological mediator of inflammation and also plays a central role in the regulation of immune responses. A number of physiological and pharmacological agents, such as PGE2, IL-1{alpha}, histamine, {beta}2-adrenergic receptor agonists, and phosphodiesterase inhibitors, share the ability to elevate intracellular cAMP in T lymphocytes. Activation of the protein kinase A pathway has a variety of transcriptional and posttranscriptional effects on different immune mediators. We have reported that increased levels of cAMP have an important posttranscriptional effect on the regulation of CD40 ligand (16). It has also been shown that elevation of intracellular cAMP promotes IL-10 secretion and Th2 activation while it inhibits T cell proliferation, IL-2 and IL-12 production, and Th1-mediated effector functions (17, 18, 19, 20, 21). In the present study we show that TCR stimulation of naive CD4+ T cells in the presence of cAMP-elevating agents leads to a population of CD4+CD45RA+ T cells with functional and phenotypic features of effector Th2 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PMA, ionomycin, dibutyryl cAMP (dbcAMP),3 PGE2, recombinant human IL-2 (rhIL-2), mitomycin C, and p-nitrophenyl phosphate disodium saltwere obtained from Sigma-Aldrich (St. Louis, MO). The mAbs CD45RA-FITC, CD2-FITC, CD11a-FITC, CD44-FITC, HLA-DR-FITC, CD30-FITC, CD62L-PE, CD45RO-PE, CD25-PE, CD71-PE, CD3-PerCP, CD4-PerCP, CD56, and isotype-matched irrelevant controls were from BD Biosciences (San Jose, CA). rhIL-4, rhIL-10, and rhIL-13 and neutralizing mAb to IL-4, IL-10, and IL-13 were purchased from R&D Systems (Minneapolis, MN). Purified and biotinylated anti-human IL-10 mAb were purchased from BD PharMingen (San Diego, CA). Mouse anti-CD45RO and anti-CD45RA mAb and streptavidin-alkaline phosphatase conjugate were purchased from Caltag Laboratories (San Francisco, CA).

Cell preparation

Blood was obtained from healthy adult volunteers or buffy coats obtained from routine blood donors (Community Transfusion Center, Oviedo, Spain) or from the placental segment of the umbilical cords of normal full-term deliveries. Mononuclear blood cells were obtained from all samples by centrifugation over Ficoll-Hypaque gradients (Lymphoprep, Nycomed, Oslo, Norway). Cord blood samples were subjected to a second round of Ficoll-Hypaque density centrifugation to remove contaminating immature RBC. T lymphocytes were further purified by negative depletion with anti-CD14 plus Pan B Dynabeads (Dynal Biotech, Oslo, Norway), obtaining a purity >96% as determined by flow cytometry. CD4+ T lymphocytes were isolated from mononuclear cells by complement-mediated negative selection using Th Lympho-kwik (One Lambda, Canoga Park, CA) plus CD56 mAb to eliminate NK cells. The purity of CD4+ T cells obtained was >98%, as determined by flow cytometry. For CD4+CD45RA+ or CD4+CD45RO+ T cell purification, 107 CD4+ T cells/ml were incubated for 30 min in RPMI and 5% FCS (ICN, Flow, Costa Mesa, CA) with saturating amounts of mouse anti-CD45RO or anti-CD45RA mAb, respectively. The two different CD4+ T cell subsets were isolated by negative depletion after incubation with goat anti-mouse IgG Dynabeads followed by magnetic separation. The purity obtained was >97%, as determined by flow cytometry.

Cell activation and culture conditions

Adult and neonatal mononuclear blood cells or purified T cell subsets were cultured at a concentration of 2 x 106 cells/ml at 37°C in 5% CO2 in medium RPMI 1640 containing 2 mM L-glutamine and 25 mM HEPES (BioWhittaker, Verviers, Belgium) and supplemented with 10% heat-inactivated FCS and antibiotics. Cell activation was set up in a 1-ml volume in 24-well culture plates previously coated with activating mAb. Wells were incubated overnight at 4°C with 0.5 ml PBS containing 25 µg/ml anti-CD3 (OKT3; Ortho, Raritan, NJ) and 2.5 µg/ml anti-CD28 (Serotec, Oxford, U.K.) mAb. Before use, wells were washed twice with PBS and then with culture medium. When indicated, PGE2 and dbcAMP were added to cultures at concentrations of 1 µM and 0.5 mM, respectively.

Flow cytometry

The expression of CD45 isoforms and phenotypic studies of cultured cells were performed after two- or three-color staining with the appropriate mAb and were analyzed using a FACScan flow cytometer (BD Biosciences). A minimum of 10,000 lymphocyte-gated events were acquired and analyzed with CellQuest software (BD Biosciences). The specific fluorescence intensity was quantified as the mean fluorescence intensity calculated by subtracting the background of isotype-matched control staining from the total fluorescence.

Determination of apoptosis

Detection of apoptosis in cultured cells was made using FlowTACS In Situ TUNEL-Based Apoptosis Detection kit (R&D Systems), performed according to the manufacturer’s instructions. In situ DNA fragmentation at the single-cell level was quantified by flow cytometric detection of the biotinylated nucleotides enzymatically added to the 3' ends of the DNA fragments. Results are expressed as the percentage of positive stained cells.

Proliferation assays

Cellular proliferation was determined quantifying [3H]thymidine incorporation to cultured cells. Triplicate cultures were conducted in 96-well round-bottom microtiter plates with the stimuli indicated in each experiment. After 72 h, cultures were pulsed with 1 µCi/well [3H]thymidine (ICN) for 8 h, and cells were harvested onto glass-fiber filters for counting cell-incorporated thymidine using standard scintillation techniques (Packard Instruments, Downers Grove, IL). Results are expressed as the mean counts per minute ± SD for triplicate cultures.

Mixed leukocyte reaction

Peripheral blood T cells were stimulated with immobilized anti-CD3 and anti-CD28 mAb in the presence or the absence of dbcAMP. After 6 days of culture, cells were treated with 25 µg/ml mitomycin C (stimulator cells) and cocultured with peripheral blood mononuclear responder cells from HLA-mismatched individuals. Triplicate cultures of 1 x 105 responder cells/well and 0.25–2 x 105 stimulator cells/well in 200 µl complete culture medium were conducted for 5 days, pulsed with 1 µCi/well [3H]thymidine for the last 12 h of the assay, and harvested, and [3H]thymidine incorporation was counted as described for proliferation assays.

Cytokine production

Adult and neonatal mononuclear cells or purified CD4+ T cell subsets were cultured in mAb-coated plates for the time indicated, and the cytokine concentration in culture supernatants was determined by ELISA techniques. IL-4, IL-13, TNF-{alpha}, and IFN-{gamma} were quantified using commercial mAb sandwich ELISA kits (Quantikine; R&D Systems), performed according to the manufacturer’s instructions. An in-house ELISA was employed to quantify IL-10, as follows. Microtiter wells were coated overnight with affinity-purified anti-human IL-10 mAb (R&D Systems) and blocked with 1% casein in TBS for 2 h at 37°C. Samples and IL-10 standards (R&D Systems) were diluted in blocking solution and incubated for 18 h at 4°C. After washing with TBST (0.05%), wells were incubated for 2 h with biotinylated anti-human IL-10 mAb (R&D Systems), washed, incubated for 1 h with streptavidin-alkaline phosphatase conjugate, and revealed using p-nitrophenyl phosphate disodium salt as substrate. Absorbance was determined at a wavelength of 405 nm. Quantities of IL-10 were calculated according to the standard curves. The lower limit of detection was 0.25 pg/ml for IL-4, 31.2 pg/ml for IL-13, 15.6 pg/ml for IFN-{gamma}, 4.4 pg/ml for TNF-{alpha}, and 7.5 pg/ml for IL-10.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cAMP-elevating agents prevent phenotypic conversion to CD45RO in activated CD4+ T cells

Adult or neonatal mononuclear cells were stimulated with plate-bound anti-CD3 and anti-CD28 mAb in the presence or the absence of cAMP-elevating agents (dbcAMP and PGE2). After 6 days of culture, cells were harvested, and the surface expression of the CD45 isoforms was analyzed by flow cytometry. Upon anti-CD3 and anti-CD28 activation, most adult cells acquire the CD45RO phenotype, characteristic of primed/memory T cells (Fig. 1GoA). In contrast, cells stimulated in the presence of PGE2 or dbcAMP remain mostly CD45RA+, indicating that these agents prevent acquisition of the CD45RO phenotype in CD3/CD28-activated T cells. The majority of neonatal cells freshly obtained from cord blood express the CD45RA+RO--unprimed phenotype (Fig. 1GoB). A large percentage of these cells suffer the splicing and express the CD45RO Ag upon activation with anti-CD3 and anti-CD28 mAb. However, similar to adult lymphocytes, the presence in the cell cultures of cAMP-elevating agents impedes acquisition of the memory phenotype. Similar results were obtained with 10-3 M teophylline and 25 µg/ml adrenaline (data not show).



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  		FIGURE 1. cAMP-elevating agents prevent the acquisition of the CD45RO+ phenotype. Adult (A) and neonatal (B) mononuclear blood cells were cultured for 6 days in medium alone or with plate-bound anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP (0.5 mM) and PGE2 (1 µM). The histograms represent 10,000 CD3+ gated lymphocytes stained for CD45RO and analyzed using a FACScan flow cytometer with CellQuest software. The percentage of positively stained cells is indicated in each histogram. Profiles are from a single representative experiment of five independent experiments.

 
To know whether dbcAMP was able to revert the CD45RO+ phenotype, CD4+CD45RA+ and CD4+CD45RO+ T lymphocytes were isolated from adult peripheral blood and cultured with immobilized anti-CD3 and anti-CD28 mAb with or without dbcAMP (Fig. 2Go). As expected, CD4+CD45RA+ T cells changed to express the CD45RO isoform following activation, while the switch was prevented in the presence of dbcAMP. However, purified CD4+CD45RO+ T lymphocytes continued expressing the CD45RO Ag independently of the presence of dbcAMP in the culture medium, indicating that this agent was unable to reverse the memory phenotype. Naive CD4+ T cells primed in the presence of cAMP-elevating agents and expressing the CD45RA+ phenotype are referred to as cAMP-primed CD45RA cells.



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  		FIGURE 2. dbcAMP does not reverse the CD45RO phenotype. Purified CD4+CD45RA+ and CD4+CD45RO+ T lymphocytes were stimulated for 6 days with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP and stained for flow cytometric analysis. Overlay histograms represent CD45RO fluorescence intensity after 6 days of stimulation. Histograms are representative of two independent experiments.

 
We further studied the occurrence of activation-induced apoptosis in T cells and its modulation by cAMP-elevating agents. In situ DNA fragmentation was measured using a TUNEL-based procedure. A considerable degree of apoptosis was induced by cell activation with anti-CD3 and anti-CD28 (28.46% of apoptotic cells), whereas the presence of dbcAMP inhibited the anti-CD3- and anti-CD28-induced apoptosis, suggesting that it is a survival factor for primed CD45RA+ T cells (Fig. 3Go).



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  		FIGURE 3. dbcAMP inhibits apoptosis in activated T lymphocytes. PBMCs were stimulated for 6 days with anti-CD3 and anti-CD28 mAb in the absence or the presence of dbcAMP. The occurrence of apoptosis was determined at the single-cell level by flow cytometric quantification of the stained nucleotides enzymatically added to the 3' end of the DNA fragments (TUNEL-based method). The percentage of positively stained cells is represented in each histogram. Data are from one of three independent experiments with comparable results.

 
Inhibition of CD45 splicing by dbcAMP is independent of IL-2

Since cAMP-elevating agents inhibited the production of IL-2, we analyzed the role of this lymphokine in the cAMP-dependent inhibition of CD45 splicing. Addition of 200 U/ml rhIL-2 to T lymphocytes cultured in the presence of anti-CD3, anti-CD28, and dbcAMP greatly reverted the cAMP-induced inhibition of cell proliferation (Fig. 4GoA). However, this cytokine showed limited capacity to reverse the dbcAMP-dependent prevention of the splicing toward CD45RO phenotype (Fig. 4GoB). Similar results were obtained with 100–500 U/ml rhIL-2 (data not shown). These results suggest that the effect of dbcAMP on CD45 splicing is not only dependent on its capacity to inhibit cell proliferation. Moreover, cells previously activated for 6 days with either anti-CD3 and anti-CD28 or anti-CD3, anti-CD28, and dbcAMP responded to restimulation with ionomycin plus PMA by growing, indicating that cells primed in the presence of dbcAMP are not anergized, as they maintain their proliferative capacity similarly to cells primed in its absence (Fig. 5Go).



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  		FIGURE 4. IL-2 does not reverse dbcAMP inhibition of CD45 splicing. Peripheral blood T cells were cultured for 6 days with the stimuli indicated. Cellular proliferation is represented as the mean and SD of [3H]thymidine uptake from triplicate cultures (A). The percentage of CD4+ T lymphocytes expressing the CD45RO isoform was determined by flow cytometric analysis (B). rhIL-2 was added at 200 U/ml. Results are representative of three independent experiments.

 


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  		FIGURE 5. cAMP-primed CD45RA cells proliferate in response to mitogen stimulation. Peripheral blood T cells were cultured with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP. After 6 days, primed cells were washed and recultured in medium alone or with ionomycin (1 µg/ml) and PMA (10 ng/ml) for 3 days. Cellular proliferation was measured by quantifying [3H]thymidine incorporation. Results are shown as the mean ± SD of triplicate cultures and are representative of two independent experiments.

 
Surface Ag expression in cAMP-primed-CD45RA lymphocytes

Next we studied whether cAMP-primed CD45RA lymphocytes expressed surface molecules characteristics of effector/memory cells. Purified CD4+CD45RA+ peripheral blood T cells were incubated with immobilized anti-CD3 and anti-CD28 mAb with or without dbcAMP for 6 days. Following this period, the cell surface expression of adhesion molecules (Fig. 6Go) and activation Ags (Fig. 7Go) were analyzed by flow cytometry. Naive CD4+ T cells cultured in the presence of anti-CD3, anti-CD28, and dbcAMP significantly up-regulated the expression of CD2, CD11a, and CD44 molecules over that obtained in the presence of medium or dbcAMP alone. The surface marker L-selectin (CD62L), which is typically shed from T lymphocytes when undergoing transition from naive to memory cells, was down-regulated in cAMP-primed CD45RA cells. The study of the expression of activation Ags (Fig. 7Go) yielded interesting results. Naive CD4+ T cells activated with anti-CD3, anti-CD28, and dbcAMP expressed higher levels of CD30, CD71, and HLA-DR molecules than lymphocytes activated in the absence of dbcAMP. The expression of CD25 was also up-regulated in cells activated by the CD3/CD28 pathway and dbcAMP, although in this case the level of surface expression was lower than that obtained in cells stimulated in the absence of dbcAMP. In addition, cells primed in the presence of dbcAMP are more efficient stimulators in allogenic responses than cells primed in its absence, indicating the physiological relevance of cAMP-induced up-regulation of HLA-DR molecules in T cells (Fig. 8Go). All these results together indicate that cAMP-primed CD45RA cells are truly activated lymphocytes



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  		FIGURE 6. Expression of adhesion molecules in cAMP-primed CD45RA cells. Purified CD4+CD45RA+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP for 6 days. The histograms represent 10,000 cells stained with the indicated mAb (filled histogram) or isotype-matched control (dotted lines). Numbers are the mean fluorescence values obtained with the indicated mAb minus negative control staining. Results are representative of four independent experiments.

 


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  		FIGURE 7. Expression of activation markers in cAMP-primed CD45RA cells. Purified CD4+CD45RA+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP for 6 days. The histograms represent 10,000 cells stained with the indicated mAb (filled histograms) or isotype-matched control (dotted lines). Numbers are the mean fluorescence values obtained with the indicated mAb minus negative control staining. Results are representative of four independent experiments.

 


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  		FIGURE 8. cAMP-primed CD45RA cells are very efficient stimulator cells in an MLR. Peripheral blood T cells were stimulated for 6 days with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP. At the end of the culture period cells were treated with 25 µg/ml mitomycin C, and different concentrations were cocultured with 105 allogenic PBMC from HLA-mismatched individuals. Cellular proliferation was measured by quantifying [3H]thymidine incorporation. Results are shown as the mean ± SD of triplicate cultures and are representative of two independent experiments.

 
Cytokine production by cAMP-primed CD45RA lymphocytes

The possible effector functions of cells primed in the presence of cAMP-elevating agents were analyzed by studying the profile of the cytokines secreted. The cytokine protein levels obtained after 6 days of anti-CD3 and anti-CD28 stimulation in the presence or the absence of dbcAMP were quantified by ELISA in the supernatants of PBMC (Fig. 9Go). The presence of dbcAMP in the cell cultures induced the production of high levels of IL-4, IL-10, and IL-13, while negligible amounts of IFN-{gamma} and TNF-{alpha} were synthesized. This profile of cytokine production corresponded to lymphocytes with the Th2 phenotype. In contrast, cells activated with anti-CD3 and anti-CD28 alone were high producers of IFN-{gamma} and TNF-{alpha}. These results support the hypothesis that the presence of cAMP-elevating agents at priming predisposes T cells to differentiate toward a Th2 phenotype.



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  		FIGURE 9. Cytokine production by cells stimulated in the presence of dbcAMP. PBMC were cultured for 6 days with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP. Culture supernatants were harvested and assayed for IL-4, IL-10, IL-13, IFN-{gamma}, and TNF-{alpha} production by ELISA techniques. The mean and SD of four independent experiments are shown.

 
The capacity of cAMP-primed CD45RA lymphocytes to produce a Th2 pattern of cytokines was confirmed by studying neonatal lymphocytes and adult purified CD4+CD45RA+ and CD4+CD45RO+ T cell subsets. Confirming the above results, Th2 cytokines (IL-4, IL-10, and IL-13) were readily synthesized by neonatal (Fig. 10GoA) and adult CD4+CD45RA+ (Fig. 10GoB) lymphocytes stimulated in the presence of dbcAMP (cAMP-primed CD45RA cells), while the highest amount of the Th1 cytokine IFN-{gamma} was secreted by effector CD4+CD45RO+ T cells generated by stimulation of CD4+CD45RA+ adult cells in the absence of dbcAMP (Fig. 10GoB). These results indicate that cAMP-primed CD45RA lymphocytes have effector characteristics of Th2 cells. Purified CD4+CD45RO+ T cells (Fig. 10GoC) secrete considerable amounts of IL-4, IL-10, and IFN-{gamma} after anti-CD3 and anti-CD28 stimulation, whereas only a small concentration of IL-4 was produced when dbcAMP was present. Kinetic studies performed at earlier times after activation (Fig. 11Go) showed that naive and memory T cells display the same pattern of kinetic expression of IL-13 and IFN-{gamma} following CD3/CD28 stimulation, while memory cells produce a higher concentration of IL-10 than naive cells with the same stimuli. The kinetic pattern of cytokine production changed upon addition of dbcAMP. Secretion of cytokines by memory cells was highly diminished, whereas IL-10 and IL-13 production by naive cells was considerably up-regulated. IFN-{gamma} production was inhibited in both cell types by dbcAMP. Thus, the effect of dbcAMP is clearly different in naive and memory cells, being mainly relevant in the high production of IL-13 by cAMP-primed CD45RA cells.



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  		FIGURE 10. cAMP-primed CD45RA cells secrete Th2 cytokines. Neonatal lymphocytes (A) or purified adult CD4+CD45RA+ (B) or CD4+CD45RO+ (C) T cells were cultured for 6 days with anti-CD3 and anti-CD28 mAb in the presence or the absence of dbcAMP. Culture supernatants were harvested and assayed for IL-4, IL-10, IL-13, and IFN-{gamma} production by ELISA techniques. The mean and SD of four umbilical cord and three adult donors are shown.

 


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  		FIGURE 11. Effect of dbcAMP on cytokine production by naive and memory cells. Purified CD4+CD45RA+ and CD4+CD45RO+ T cells were cultured for 1, 2, 3, and 6 days with anti-CD3 and anti-CD28 mAb in the presence or the absence of dbcAMP. Culture supernatants were harvested and assayed for IL-10, IL-13, and IFN-{gamma} production by ELISA techniques. Kinetics are representative of three independent experiments.

 
Since cAMP-primed CD45RA cells secrete immunosupressor cytokines, we hypothesized whether endogenous production of these molecules could cause the cAMP-induced inhibition of CD45 splicing. To verify this, we added either IL-4, IL-10, or IL-13 to anti-CD3- and anti-CD28-activated lymphocytes, and the expression of CD45RO Ag was analyzed 6 days later. Fig. 12Go shows that IL-4 and IL-10 partially inhibited phenotypic conversion to CD45RO, although to a much lesser extent than cAMP. IL-13 had no effect; this was probably due to the lack of IL-13R on the membrane of T lymphocytes. We also blocked the endogenous production of IL-4 and IL-10 by adding different amounts (5–100 µg/ml) of neutralizing anti-IL-4 and anti-IL-10 mAb to cells activated by CD3/CD28 and dbcAMP, obtaining similar results when a concentration of 20 µg/ml or higher was employed. Anti-IL-4 mAb had no effect in reverting the dbcAMP-induced inhibition of CD45 splicing, whereas a slight effect was observed with anti-IL-10, indicating that these cytokines are not responsible for the cAMP-dependent inhibition of CD45 splicing.



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  		FIGURE 12. Effects of immunosupressor cytokines on CD45RO phenotype acquisition. Peripheral blood T cells were stimulated with anti-CD3 and anti-CD28 in the presence or the absence of dbcAMP and the cytokines (100 ng/ml) or the neutralizing mAb (40 µg/ml) indicated. After 6 days of culture cells were harvested, and CD45RO surface expression was analyzed by flow cytometry in 10,000 CD4+ gated cells. Results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Surface expression of the CD45 Ag has been widely used to define the pool in which naive or memory/effector cells reside. After TCR cross-linking, naive T cells lose the CD45RA isoform and switch to the CD45RO phenotype, acquiring effector function capabilities. In the present report we show that the presence of cAMP-elevating agents during anti-CD3 and anti-CD28 activation of naive CD4+ T cells prevents CD45RO expression and leads to a population of CD4+CD45RA+ lymphocytes with functional and phenotypic characteristics different from "truly" naive T cells. In fact, these cAMP-activated cells display on their membranes a pattern of adhesion molecules and activation Ags characteristic of primed cells, secrete substantial amounts of certain cytokines, and are very efficient stimulator cells in a MLR. We have called these cells cAMP-primed CD45RA lymphocytes.

It is well known that high levels of intracellular cAMP inhibit IL-2 transcription, which, in turn, is responsible for the low rate of proliferation displayed by cells cultured with dbcAMP (20, 22). It may be possible that the lack of proliferation and IL-2 production by dbcAMP-activated cells causes the CD45 splicing inhibition. However, we do not think this is the case, since addition of exogenous IL-2 highly incremented cell proliferation and the expression of IL-2R{alpha} (data not shown), whereas the surface expression of CD45RO Ag was only slightly modified. One important feature of cAMP-primed CD45RA lymphocytes is that they have a prolonged survival compared with CD45RO+ effector cells, since the presence of dbcAMP in the cell cultures prevented the anti-CD3- plus anti-CD28-induced apoptosis. In accordance with this, it has been reported that PGE2 inhibited T cell activation-induced apoptosis in murine splenic T cells by down-regulating Fas ligand mRNA levels (23). Furthermore, the presence of pentoxifylline, a phosphodiesterase inhibitor, during primary alloantigen exposure of T cells avoided apoptosis and incremented the secondary proliferative responses (24).

Naive CD4+ T cells activated via CD3/CD28 and dbcAMP showed a pattern of adhesion molecules comparable to that of CD4+CD45RO+ effector T cells obtained by activation without dbcAMP. In both cell types a down-regulation of L-selectin (CD62L) was observed, probably related to the reduced ability of memory/effector lymphocytes for lymph node recirculation compared with naive T cells. Accordingly, a significant decrease in the number of CD4+CD62L+ circulating T cells has been reported in response to isoproterenol infusion (a {beta}2-adrenergic agonist) in healthy subjects (25). The surface level of other adhesion molecules (CD2, CD11a, and CD44) was up-regulated in cAMP-primed CD45RA lymphocytes, although to a lesser extent than in CD3/CD28-activated cells in the absence of dbcAMP. Activation markers were also up-regulated in cAMP-primed CD45RA cells and, with the exception of CD25 Ag, the levels of surface expression of these molecules were higher than those displayed by CD4+CD45RO+ T cells obtained after lymphocyte stimulation via CD3/CD28 without dbcAMP. The activation marker CD30, mainly expressed on the membrane of Th2 effector cells (26), was significantly represented in cAMP-primed CD45RA lymphocytes. This finding is in agreement with the Th2 cytokine profile secreted by these cells as discussed below. The effect of dbcAMP on the expression of HLA class II Ags was of particular interest and is reported here for the first time. HLA-DR molecules were significantly up-regulated when dbcAMP was added to the CD3/CD28-stimulated cell cultures. In accordance with this observation, we found that cAMP-primed CD45RA cells were very efficient stimulators in a mixed leukocyte culture. The induction of class II Ags expression by cAMP-elevating agents explains the previous observation by Gupta et al. (24), who noticed enhanced secondary alloproliferative responses when pentoxifylline was present at priming.

Naive CD4+ T lymphocytes primed in the presence of dbcAMP secreted high amounts of IL-4, IL-10, and IL-13, whereas the production of IFN-{gamma} was very low. This pattern of cytokine production corresponded to a Th2 profile of effector lymphocytes. In contrast, CD4+CD45RO+ lymphocytes generated by CD3/CD28 stimulation of CD4+CD45RA+ T cells in the absence of dbcAMP were found to secrete mainly IFN-{gamma} (Th1 phenotype). It is of interest that isolated CD4+CD45RO+ T cells stimulated in the presence of dbcAMP did not produce significant amounts of these cytokines, even when levels were measured early after initiating cell cultures. This finding is in accordance with the PGE2-induced suppression of CD4+CD45RA-ROhigh T cell clones previously described (27). The possibility that elevated levels of intracellular cAMP induce anergic signals on memory cells should be addressed in further studies. The high concentration of IL-13 secreted by both neonatal and adult CD4+CD45RA+ cells upon stimulation with dbcAMP and CD3/CD28 mAbs is particularly remarkable. Thus, we have reported a special sensitivity of neonatal cells to signals delivered by the protein kinase A pathway activation, as these cells expressed high levels of CD40 ligand and secreted significant concentrations of Th2 cytokines (IL-4 and IL-10) in response to ionomycin and cAMP-elevating agents (28). The presence in the feto-placental environment of PGE2, progesterone, and other uterine hormones that increase intracellular cAMP levels (29) may induce the secretion of immunosupressor cytokines, inhibiting Th1 differentiation, which could lead to loss of pregnancy (30). Also, it may explain the reduced incidence and severity of graft-vs-host disease with transplanted cord blood compared with adult bone marrow cells (31, 32).

Since cAMP-primed CD45RA lymphocytes produce cytokines with immunosupressor abilities, we hypothesized whether these proteins were responsible for the inhibition of CD45 splicing upon activation with dbcAMP. We answered this question by supplementing with exogenous IL-4 or IL-10 the CD3/CD28 cultures and by neutralizing the endogenous production of these molecules with anti-IL-4 or anti-IL-10 mAb in the cultures stimulated in the presence of dbcAMP. Employing these two experimental approaches we concluded that the inhibition of CD45 splicing by cAMP-elevating agents was only slightly affected by the actions of these two cytokines and, therefore, other unknown mechanisms may be implicated in preventing CD45RO expression in cAMP-activated cells. It will be of interest to study the role of dbcAMP in regulation of the different proteins of the SR family of splicing factors that has been recently implicated in the alternative splicing of CD45 Ag, leading to either exon inclusion or skipping (3, 33).

It is now becoming clear that in normal adults, within the pool of CD45RA+RO- T lymphocytes there are different subpopulations derived from diverse sources and performing different functions. The existence of a subset of Ag-specific CD8+CD45RA+ effector T cells is well documented (10, 15, 34, 35, 36, 37). Similarly, a subpopulation of primed CD4+ CD45RA+ cells may be revertant memory T cells, delivered from the CD45RO+ subset, which in the absence of Ag convert to quiescent, resembling naive T cells with increased Ag-specific precursor frequency and with the capacity for long life (9, 38). It has also been described that a combination of cytokines may expand and prime naive T lymphocytes to express enhanced levels of adhesion and activation molecules while retaining the CD45RA+RO- phenotype (11, 12, 13, 39). Unutmaz et al. (11) reported that these cells produce considerable amounts of cytokines (IL-3/GM-CSF, IL-6, TNF-{alpha}, and IFN-{gamma}) when stimulated with anti-CD3 mAb in the absence of accessory cells. These lymphocytes, however, do not express HLA class II Ags and do not secret Th2 cytokines; therefore, they are different from the cAMP-primed CD45RA cells described in this report.

Our results support the previous findings that in several in vivo situations CD4+ CD45RA+ T lymphocytes may exert important effector functions (40, 41, 42, 43). In atopic patients a quantitative and qualitative defect in CD4+CD45RO+ T cells has been reported, whereas CD4+CD45RA+ T lymphocytes secreted IL-4 and IL-5 and could promote IgE and IgA production. The frequency of cells responding to allergens within the CD45RA subset was 7- to 20-fold higher than that in nonatopic patients (44, 45). Activated CD4+CD45RA+ T lymphocytes were also the prevailing inflammatory cell population in discoid lupus and children with nephrotic syndrome (46, 47, 48). As atopic and inflammatory responses lead to liberation of cAMP-increasing agents, such as PGE2, IL-1{alpha}, and histamine, the CD4+CD45RA+ effector cells found in these conditions may result from Ag stimulation of naive cells in the presence of these mediators.


   Footnotes
 
1 This work was supported by Grant 00/150 from Fondo de Investigaciones Sanitarias de la Seguridad Social. Back

2 Address correspondence and reprint requests to Dr. Carmen Gutiérrez, Servicio de Inmunología, Hospital Central de Asturias, Julián Clavería s/n, 33006 Oviedo, Spain. E-mail address: cguti{at}hca.es Back

3 Abbreviations used in this paper: dbcAMP, dibutyryl cAMP; rhIL-2, recombinant human IL-2. Back

Received for publication October 18, 2001. Accepted for publication May 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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